**Background:** The circadian clock, driven by core clock genes such as BMAL1, regulates numerous cellular processes including proliferation, DNA repair, and redox state. BMAL1 and CLOCK form a heterodimer that promotes transcription of clock repressors (PER, CRY) and also influences chromatin remodeling through histone modifications. While BMAL1 is known to be important for skin homeostasis, hair cycle, and wound healing, its role in epigenetic modifications in the epidermis, particularly histone acetylation, is poorly understood. This study aimed to characterize BMAL1-driven histone acetylation changes in epithelial cells during homeostasis and in response to injury.
**Methods:** The authors used a loss-of-function approach in vivo with Bmal1 knockout mice (Bmal1-/-; n=5, male) and wild-type (WT) controls (n=4, male). Full-thickness wounds were created on the dorsal skin of 4-6-week-old mice using a 5-mm punch biopsy, and silicone rings were sutured around the wounds to prevent contraction. Tissues were collected at post-operative day 5 at Zeitgeber time 2 (ZT2). Immunofluorescence staining was performed on paraffin-embedded sections using antibodies against acetylated histones: H3 Lys9 (H3K9), H3 Lys27 (H3K27), H4 Lys5 (H4K5), H4 Lys8 (H4K8), and H4 Lys16 (H4K16). The histone labeling index (percentage of positive cells) was quantified from at least three independent fields using ImageJ. Statistical analysis used t-tests with significance at p ≤ 0.05.
**Key Results:** During homeostasis, Bmal1-/- skin showed significant hyperacetylation of H3K9 (43.11 ± 3.11% vs. 14.76 ± 1.73% in WT; p ≤ 0.0001) and H4K8 (29.63 ± 4.14% vs. 13.45 ± 1.29%; p = 0.0002), and deacetylation of H4K16 (8.99 ± 1.41% vs. 15.16 ± 1.15%; p = 0.005). No significant changes were seen for H3K27 or H4K5. In injured skin, Bmal1 ablation primarily affected histone H4: hyperacetylation of H4K5 (45.07 ± 3.57% vs. control), H4K8 (15.92 ± 2.64%), and H4K16 (19.70 ± 1.34%) in the epidermis (p ≤ 0.05 to p ≤ 0.0001). In the dermis, Bmal1-/- mice showed decreased H3K27 (42.79 ± 2.83%) and H4K8 (38.23 ± 3.06%) acetylation, and hyperacetylation of H4K5 (51.88 ± 1.28%). In the wound bed, hyperacetylation of H4K5 (26.48 ± 1.54%) and H4K16 (35.59 ± 2.02%) was observed (p ≤ 0.01 to p ≤ 0.0001). At the wound margin, Bmal1-/- cells displayed hypoacetylation of H3K9 (p ≤ 0.05) and hyperacetylation of H4K8 and H4K16 (p ≤ 0.001). In the migratory epithelial tongue, only H4K5 was significantly hyperacetylated (p ≤ 0.0001). Overall, Bmal1 deletion led to widespread H4 hyperacetylation during wound healing, with site-specific changes in H3 acetylation.
**Clinical Implications:** This study demonstrates that BMAL1 is a key regulator of histone acetylation in the skin, both at baseline and during wound repair. The observed hyperacetylation of histones H4 in Bmal1-deficient mice correlates with delayed wound healing, suggesting that proper BMAL1 function is necessary for timely regeneration. The findings highlight the potential of targeting epigenetic modifications, such as using HDAC inhibitors (e.g., valproic acid), to modulate histone acetylation and improve wound healing. However, the small sample size (n=4-5 per group) is a limitation, and further studies with larger cohorts are needed to confirm these results and explore therapeutic applications.